PROJECT SUMMARY/ABSTRACT Duchenne Muscular Dystrophy (DMD) is a fatal X-linked childhood skeletal muscle degenerative disease. Affected children become non-ambulatory within the first decade of their lives and die within the third. There is no effective treatment available that can either cure or stop the progression of DMD. Lack of a detailed understanding of the key myogenic processes and DMD pathophysiology at the molecular level significantly hinders the development of an effective therapy. The goal of this research proposal is to unravel the role of N6- methyl-adenosine (m6A) methylation in skeletal muscle differentiation, regeneration, and DMD pathophysiology. m6A is the most abundant and reversible epitranscriptomic mark in eukaryotic mRNAs. m6A methylation is implicated in differentiation and development and is dysregulated in numerous human diseases. m6A mark is catalyzed by methyltransferase-like 3 (Mettl3) and is removed by the RNA demethylase enzymes, fat mass- and obesity-associated (FTO) and alkylation repair homolog 5 (ALKBH5). A recent study dismisses FTO as an m6A demethylase but the function of FTO is implicated in body mass regulation and myogenesis. However, it remains completely unknown whether Mettl3 has any role in skeletal muscle function. We have found that Mettl3 and m6A levels are downregulated during myoblast differentiation and muscle regeneration. Knockdown of Mettl3 decreases m6A level and promotes skeletal muscle differentiation and regeneration. Our data further show that Mettl3 controls myogenesis by directly installing m6A methylation marks near the stop codons of Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1?) and destabilizing PGC-1? transcripts. PGC-1? is a master regulator of mitochondrial biogenesis pathways that play a critical role in myogenesis and DMD. We have found that the Mettl3/PGC-1? axis is dysregulated in both satellite cells and myofibers isolated from mdx mouse model of DMD. We hypothesize that m6A mRNA methylation regulates skeletal muscle function and an elevated m6A level has implication in DMD pathophysiology. For the first time, we will demonstrate the role of m6A methyltransferase and m6A mRNA modification in skeletal muscle function and establish the underlying molecular mechanism by which m6A methylation regulates the key myogenic processes. We will examine the role of m6A methylation in a clinically relevant mdx mouse model system to dissect the function of m6A in DMD pathophysiology. Our proposed research will make a significant impact by elucidating a fundamental epitranscriptomic mechanism implicating skeletal muscle differentiation, regeneration, and DMD pathophysiology, and also by revealing a new therapeutic axis for DMD.